Human embryonic stem cell-derived oligodendrocyte progenitors aid in functional recovery of sensory pathways following contusive spinal cord injury

Abstract

Background: Transplantations of human stem cell derivatives have been widely investigated in rodent models for the potential restoration of function of neural pathways after spinal cord injury (SCI). Studies have already demonstrated cells survival following transplantation in SCI. We sought to evaluate survival and potential therapeutic effects of transplanted human embryonic stem (hES) cell-derived oligodendrocyte progenitor cells (OPCs) in a contusive injury in rats. Bioluminescence imaging was utilized to verify survivability of cells up to 4 weeks, and somatosensory evoked potential (SSEPs) were recorded at the cortex to monitor function of sensory pathways throughout the 6-week recovery period.

Principal findings: hES cells were transduced with the firefly luciferase gene and differentiated into OPCs. OPCs were transplanted into the lesion epicenter of rat spinal cords 2 hours after inducing a moderate contusive SCI. The hES-treatment group showed improved SSEPs, including increased amplitude and decreased latencies, compared to the control group. The bioluminescence of transplanted OPCs decreased by 97% in the injured spinal cord compared to only 80% when injected into an uninjured spinal cord. Bioluminescence increased in both experimental groups such that by week 3, no statistical difference was detected, signifying that the cells survived and proliferated independent of injury. Post-mortem histology of the spinal cords showed integration of human cells expressing mature oligodendrocyte markers and myelin basic protein without the expression of markers for astrocytes (GFAP) or pluripotent cells (OCT4).

Conclusions: hES-derived OPCs transplanted 2 hours after contusive SCI survive and differentiate into OLs that produce MBP. Treated rats demonstrated functional improvements in SSEP amplitudes and latencies compared to controls as early as 1 week post-injury. Finally, the hostile injury microenvironment at 2 hours post-injury initially caused increased cell death but did not affect the long-term cell proliferation or survival, indicating that cells can be transplanted sooner than conventionally accepted.

Figure 1. SSEP results showing functional improvements in spinal cord injured rats following transplantation of hES-derived OPCs compared with rats receiving no treatment.

A) Representative mean SSEP sweeps from one rat from each experimental group. Left: SSEPs recorded upon hindlimb stimulation following T8 contusion; Right: internal controls due to forelimb stimulation. B) Amplitude (hindlimbs) results quantified for the two experimental groups showing a significant benefit for OPC transplantation versus no treatment. C) Latency (hindlimbs) results reveal that the OP-transplant group exhibited remyelination, as the time of the SSEP signal transduction reduces back to baseline values. This result is indicative of higher conductivity of axonal pathways, which leads to lower latencies for the OPC-transplant group. D) Latencies of forelimb SSEPs over time did not change post-injury in both groups indicating no harm to forelimb sensory pathways. *p<0.05 and **p<0.01.

Figure 2. Immunohistochemical staining showing survival and cell type profiling of transplanted cells surrounding the injury epicenter approximately two months after injury.

(A) Dual staining for human nuclear antigen (HNA, green) and myelin basic protein (MBP, red) verifies the survival of human OPCs in the rat tissue at the site of injection into the epicenter of the injury. Cells indicated by the arrow are shown at higher magnification in the insert. The majority of these cells are surrounded by intense MBP staining at the site of injection into the grey matter where MBP expression is normally low demonstrating the production of myelin in these areas. Nuclei of both the human and rat cells are identified by DAPI (blue). (B) Triple stains for HNA (blue), the astrocytic marker GFAP (green) and mature oligodendrocyte MBP (red) at these sights confirm that the majority of transplanted cells did not express the astrocyte marker GFAP. Instead astrocytic extensions (arrowhead) can be seen reaching into the area of injury from the surrounding white matter. Insert indicated by the arrow in B shows that human OPCs indicated by the blue HNA stain were primarily located at sites of intense MBP staining. (C) Staining for the pluripotent marker OCT4 (green) was not detected in the tissue.

Figure 3. Light and electron microscopy.

Sagittal sections corresponding to the center of injury approximately 2 months after injury. H&E stained paraffin sections of spinal cords from animals subjected to contusive SCI and treated with human fibroblasts (A) or hES-derived OPCs (B) Arrows indicate areas of neuron like tracts transversing the center of injury in OPC treated rats. This is consistent with LFB (blue) and cresyl violet (purple) staining which showed significantly more cavitation (dotted circles) in the human fibroblast-treated group (C) compared to the hES-derived OPC group (D). Specifically the fibroblast treated groups shows overall loss of tissue integrity with reduced LFB and cresyl violet staining, while the OPC treated group shows the presence of neurons, stained with cresyl violet surrounded by LFB staining. Transmission electron microscopy of sagittal sections of spinal cords also showed disrupted myelin for the fibroblast-treated group (E), whereas remyelination with thin, compact sheaths was observed for the hES-OPC group (F, arrowheads). Rats treated with heat-killed OPCs (G) were similar to fibroblast controls. Magnification: (A–D, G) 40x and (E, F) 5000x. These results were verified by quantitative analysis demonstrating (H) an increase in myelin staining by LFB, (I) the number of axons identified by cresyl violet and (J) by the extent of myelination demonstrated by electron microscopy in the hES-OPC group compared to controls. Error bars represent standard deviations. Asterisks denotes statistical significance between fibroblast and ES-OPC groups (P<0.00).

Figure 4. Bioluminescence of hES derived OPC-treated groups.

Bioluminescence was followed at six different time points over a period of 4 weeks. A) Images taken at each time point that depict the bioluminescent activity of the transplanted cells. B) Total flux (photons/second) measured at various time points of the OPC-treated injury and laminectomy-only groups. Error bars represent standard deviations. C) An image of an animal showing bioluminescent activity at the site of OPC transplantation. Asterisk denotes a statistical significance in ES-OPC survival at two weeks between laminectomy and injury group (p<0.05).